1. Field of the Invention
The present invention relates to a process for the production of human tissue plasminogen activator capable of activating plasminogen and also to a human rhabdomyosarcoma-derived cell strain, KYM-SF, useful for the process.
2. Description of the Related Art
Human tissue plasminogen activator is a proteolytic enzyme which is produced by endothelial cells or the like and has high affinity to fibrin contained in aggregates of coagulated blood (thrombi). It serves to activate and convert plasminogen, an inactive proenzyme, into plasmin, a thrombolytic enzyme.
Plasmin which has been produced in circulating blood binds plasmin inhibitor in the blood and is inactivated promptly. Human tissue plasminogen activator (will hereinafter be called "t-PA"), which binds fibrin in thrombi and activates plasminogen there to dissolve the thrombi, has higher specificity to the sites of thrombi than protein therapeutic agents presently available on the market, i.e., urokinase and streptokinase. Therefore, a great deal of development work is now under way on t-PA as a thrombolytic agent which may substitute for such protein therapeutic agents.
Many reports have heretofore been presented with a view toward realizing commercial production of t-PA. Developments have been attained in both techniques making use of genetic engineering and cell culture using human-derived cells. Normal cells such as human endothelial cells and human uterine cells as well as tumor cells such as human melanoma have been known as human-derived-t-PA producing cells [Rijken et al., J. Biol. Chem., 256, 7035-7041 (1981); Vetterlein et al., J. Biol. Chem., 255, 3665-3672 (1980); Wilson et al., Cancer Research, 40, 933-938 (1980)].
A number of reports have heretofore been made on the production of substances using cell culture techniques, in which established cell lines derived from normal cells or tumor cells were adopted. This also applied to the production of t-PA as appeared in the publication of many reports (Japanese Patent Application Laid-Open Nos. 28009/1982; 110625/1984; 19486/1986; 158116/1985; 158117/1985; and 259187/1985).
On the other hand, there are some reports of clinical trials on the in vivo efficacy of t-PA obtained from animal cells manipulated by genetic recombinant techniques. [F. van der Werf et al., N. Eng. Med., 310, 609-613 (1984); The TIMI study group N. Eng. Med., 312(14), 932-936 (1985); D. Collen et al., Circulation, 73(3), 511-517 (1986)]. The results of these clinical trials demonstrated the effectiveness of t-PA but also indicated a problem that massive administration would be indispensable for allowing t-PA to act effectively [D. O. Williams et al., Circulation, 73(2), 338-346 (1986)].
As a result of the development of t-PA, it has now been considered that its effectiveness as a thrombolytic agent can be influenced by the t-PA structures as represented by the single chain or double-chain molecule. Although the structure-activity relationship of tPA.has not been fully elucidated, it is considered on the basis of reports published to date that double-chain t-PA has stronger thrombolytic ability but single-chain t-PA is superior as a thrombolytic agent in view of its in vivo stability (resistance to the action of in vivo inhibitors), its higher affinity to fibrin, etc. [E. D. Sprenger et al., Blood, 69(2), 381-387 (1987); Japanese Patent Application Laid-Open No. 118717/1984].
However, the development of t-PA has heretofore been proceeded with centering around double-chain t-PA for the fact that the structure-activity relationship has not been taken into due consideration and also for the following convenience in production. Namely, cell culture making use of a serum-added medium has been needed for the production of t-PA because host cells for recombinant manipulation and established cell lines, both of which have serum requirement, are used as starting materials for its production. In the absence of protease inhibitor, such as aprotinin or the like, it was hence impossible to avoid the conversion of single-chain t-PA, which was converted by proteolytic enzymes contained in serum components, into double-chain t-PA.
The use of serum is however accompanied by problems which should be solved for commercial production.
Namely, serum is costly and accounts for a high percentage of the cost for raw materials in the culture step. It is also necessary to remove serum-derived substances (contaminating proteins, endotoxins, mycoplasmas, virus, etc.) in order to purify a substance produced by cells and to formulate it for application as a medicine or the like. As far as the production of t-PA is concerned, it has been suggested that t-PA degrading proteases and t-PA specific inhibitors present in serum affect the productivity of t-PA [E. D. Sprenger et al., Blood, 36(2), 381-387 (1987)]. Their removal is also indispensable.
As another effect which may be given on the production of t-PA by the use of serum for cell culture, it is mentioned that the conversion of produced t-PA into double-chain t-PA under the action of certain serum-derived proteases is unavoidable. This effect is certainly associated with the problems mentioned above. It is thus required for the production of single-chain t-PA to conduct the culture in a medium supplemented with an expensive inhibitory protein (aprotinin or the like) [Japanese Patent Application Laid-Open No. 158116/1985] or a costly inhibitor [Japanese Patent Application Laid-Open No. 4233/1987]. Such an additive should be eliminated from a medicine to be formulated [Yakumu Koho, 1271, 17 (Aug. 11, 1984)]. Complex steps are however required to achieve this.
It has hence been desired to develop a technique which promises more economical and efficient production of t-PA using cell culture techniques, no matter whether the cell is produced by genetic recombination or by an established cell line or the like.
There has been a delay, especially, in the development of a technique for efficiently producing single-chain t-PA. Its development has thus been desired strongly, along with a technique for its mass production.
As a promising technique to improve productivity and to gain substantial advantages, serum-free culture of animal cells has been desired for the production of t-PA.
In the course of an investigation on various t-PA producing cells, the present inventors have already isolated a t-PA producing cell strain (IFO 50030; will hereinafter be called "KYM-A") in a clone of KYM-1 isolated from human rhabdomyosarcoma (given kindly by Dr. Morimasa Sekiguchi, The Institute of Medical Science, The University of Tokyo; IFO 50085) as disclosed in Japanese Patent Application Laid-Open No. 1382/1986. A Japanese patent application (now Japanese Patent Application Laid-Open No. 158115/1985) has been filed on a production process of t-PA, which makes use of KYM-A.
Since serum is required for the culture of KYM-A, the above process is an effective production process for double-chain t-PA alone, but not for single-chain and/or double-chain t-PA, however. Due to the use of serum, a great deal of effort has been required in order to overcome such drawbacks.